Somatic embryogenesis and in vitro culture of Huperzia selago shoots as a potential source of huperzine A

Abstract

In vitro culture of the sporophytes and somatic embryogenesis in Huperzia selago is described together with the results of phytochemical analyses to determine the huperzine A content of H. selago shoots collected from plants growing in vitro and in a natural habitat. The in vitro culture was initiated from shoots of wild plants, which underwent surface and internal sterilization to eliminate the epiphytic and endophytic bacteria and fungi and were next placed on different modifications of the Murashige and Skoog medium (MS). The highest growth frequency was achieved on the MS medium at half-strength mineral salt content (1/2 MS). The same medium was used for the induction of somatic embryogenesis. Cells of the callus, which developed from the apical meristem after 3 months of incubation, transformed into somatic embryos. The stages of their further growth resembled the zygotic embryos of other Lycopodiaceae. The developing embryo had no shoot–root axis, but one that corresponded to the shoot–foot axis with the root growing at its side. The developing somatic embryo smoothly transformed into a young sporophyte with pre-leaves and numerous proper leaves and an embryonal root. HPLC–UV assay demonstrated the highest huperzine A content (3.33 mg g −1 d.w.) in the shoots of plants grown from the somatic embryos. The yield of huperzine A from plants collected from a natural habitat differed from 0.54 (shoots harvested in spring) to 1.27 mg g −1 d.w. (shoots harvested in autumn).